Peptides binding the phosphatase 2a protein

ABSTRACT

The invention relates to novel synthetic or natural E4orf4 or Bcl-2 peptides particularly useful in antitumoral, antiviral and antiparasitic treatments, said peptides being less than 30 amino acids long and binding in vitro to a phosphatase 2A protein holoenzyme or one of its subunits. The invention also relates to polynucleotides encoding the novel peptides, vectors expressing same, as well as antibodies identifying same and probes identifying transcripts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 11/107,814filed Apr. 18, 2005, allowed, which is a continuation of PCT/FR03/03018filed Oct. 13, 2003 and claims the benefit of Canadian application2,408,207 filed Oct. 16, 2002.

FIELD OF THE INVENTION

The present invention concerns peptides binding a phosphatase 2Aprotein, an important target for the control of apoptosis, in particularin cancer cells, as well as for the control of viral and parasiticinfections.

BACKGROUND OF THE INVENTION

Given the role of the peptides of the invention in modulating theactivity of the cellular protein phosphatase 2A, it is important tomention the present state of the art on protein phosphatases 2A, theirphysiological role and their interactions with some cellular, viral orparasitic proteins.

Cell physiology is controlled in part by modulation of thephosphorylation state of proteins. The phosphorylation state of cellularproteins depends on the antagonistic action of the protein kinases thatphosphorylate them and the protein phosphatases that dephosphorylatethem.

Protein phosphatases are divided into two main groups: tyrosinephosphatases and serine/threonine phosphatases. Serine/threoninephosphatases are classified into two categories depending on thespecificity of their substrate and their sensitivity to certaininhibitors, the type 1 and type 2 phosphatases (PP1 and PP2). The type 2phosphatases are again divided into different classes, includingphosphatase 2A (PP2A), phosphatase 2B (PP2B) or calcineurin whichactivity is regulated by calcium, and phosphatase 2C (PP2C) whichactivity is magnesium-dependent.

In vivo, the serine/threonine protein phosphatases PP1 and PP2A form twofamilies of many ubiquitously expressed holoenzymes. These holoenzymesare produced by the specific interaction between their catalyticsubunits (PP1c and PP2Ac) and a wide variety of regulatory subunits.Moreover, these holoenzymes are involved in targeting and/or regulationof phosphatase activity (for recent review, see Garcia A. et al., PP1 etPP2A, des ser/thr phosphatases au coeur de l'apoptose (2001) Med/Sci 17,1214-1216).

It is now known that type 2A phosphatases are very conserved throughoutevolution and are potentially activated during regulation of variousbiological processes. The PP2A enzymes have clearly been involved intranscription control, cell cycle control, and viral transformation.Moreover, the PP2As are the targets of different viral or parasiticproteins, thus suggesting a role for PP2As in host-pathogeninteractions.

The PP2As are oligomeric complexes (holoenzymes), each of said complexescomprising a catalytic subunit C and one or two regulatory subunits, (A)and (B). The structure of subunit (A) consists of 15 imperfect repeatsof a conserved 38 to 40-amino acid sequence, some subunits (A)interacting with subunits (B) and (C). Subunits (A) and (C), conservedthroughout evolution, form the base structure of the enzyme and areconstitutively expressed. By contrast, subunits (B) form a family ofregulatory proteins differentially expressed and with no commonstructure between one another (Cohen P. The structure and regulation ofprotein phosphatases. Annu. Rev. Biochem. 1989; 58:453-508). Therefore,the protein phosphatases 2A are present in vivo under two differentforms: a dimeric form (AC) and a trimeric form (ABC). Subunits (B)regulate the phosphatase activity and the specificity towards thesubstrate. The existence of multiple forms of PP2A correlates withdistinct and varying functions of the PP2As in vivo.

Recently, it has been found that different non cellular proteins, and inparticular viral and parasitic proteins, are involved in the modulationof some specific activities of protein phosphatases 2A.

Different strategies involving the PP2A are adopted by the viruses tofacilitate their replication and survival in the host cell. For example,the parainfluenza virus incorporates, in its viral particle the PKCprotein, a protein of cellular origin under the control of the PP2A.This virus can thus perturb the host proteins' phosphorylation andfacilitate its own replication (B. P. Gupta et al. Cellular proteinkinase C ζ regulates human parainfluenza virus type 3 replication. Proc.Natl. Acad. Sci. USA 1995; 92:5204-8).

Many DNA viruses with a transforming potential, such as papovaviridae oradenoviruses, as well as some retroviruses, such as the type 1 humanimmuno-deficiency virus (HIV-1), code for proteins that interactdirectly with some host PP2As. All these viruses include proteins that,even though they are structurally different from one another, interactwith some holoenzymes and modify the phosphatase activity thereof.

More particularly, the E4orf4 protein of adenoviruses binds to aheterotrimeric PP2A and, more precisely, to a regulatory subunit (B),thus leading to a decrease in transcription of JunB in the infectedcell. This effect could play an important role during viral infection byregulating the apoptotic response in infected cells. Interestingly, theinteraction between E4orf4 and PP2A induces apoptosis of transformedcells in a p53-independent manner (Shtrichman R. et al. Adenovirus type5 E4 open reading frame 4 protein induces apoptosis in transformedcells. J. Virol. 1998; 72:2975-82).

The oncogenic DNA viruses of the Papovaridae family, including SV40 andthe polyoma virus, induce cell transformation. PP2A interacts with the“small T” antigens of SV40 and the “small T” and “middle T” of thepolyoma virus. The interactions between these viral proteins and thePP2A are clearly involved in viral transformation. Finally, thetranscriptional control, a process normally led in the cell by thedifferent factors which specifically bind to promoter regulatorysequences represents probably the most important mechanism in viralexpression control by PP2A. Therefore, PP2A is a negative regulator ofnumerous transcription factors, namely involved in the cell growth andproliferation processes, including APl/SRE, NF-kB, Spl and CREB(Waszinski, B. E. et al. Nuclear protein phosphatase 2A dephosphorylatesprotein kinase A-phosphorylated CREB and regulates CREB Transcriptionalstimulation. Mol. Cell Biol. 1993:13, 2822-34). The viral control ofthese transcription factors could allow to modulate viral transcription.

Vpr, the viral protein of HIV-1, interacts in vitro with PP2A andstimulates the catalytic activity thereof (Tung L, et al. Directactivation of protein phosphatase 2A0 by HIV-1 encoded protein complexNcp7:vpr. FEBS Lett 1997; 401: 1997-201). Vpr can induce a G2 arrest ininfected cells by inhibiting the activation of the p34cdc2-cyclin Bcomplex. In addition, Vpr interacts with the Spl transcription factorand is a weak trans-activator of the Spl-dependent transcription ofHIV-1. Therefore, the Vpr protein of HIV-1, which is incorporated withinthe virion, would be involved in vivo in the initiation of viraltranscription, an essential step in regulating the expression of the Tattranscription factor (a major regulator in the transcription coded bythe HIV-1 virus).

Unlike the protein kinases that have a well-established role inparasitic infections, serine/threonine phosphatases have only recentlybeen recognized as potentially important regulators in parasitology.

The absence of common motifs for the bulk of proteins that interact withPP2A impedes the simple identification by bio-informatics of peptidemotifs directly involved in the binding of these proteins with PP2A.

However, given the major role of protein phosphatases 2A in virus-hostor parasite-host interactions as detailed hereinabove, it will beunderstood that there is a great interest in identifying the bindingsites of viral or parasitic proteins with PP2A holoenzymes or one oftheir subunits, to identify new therapeutic targets for these viral orparasitic pathogens.

The type 1 and type 2A serine/threonine phosphatases (PP1 and PP2A)represent new potentially important targets for apoptotic control,namely in cancer cells, as well as for the control of viral or parasiticinfections (for review, see A. Garcia et al. (2000) Protein Phosphatase2A: a definite player in Viral and parasitic regulation. Microbes Inf.2,401-407; Et X. Cayla et al. (2000). La Protéine Phosphatase 2A: unenouvelle piste pour l'étude des virus et des parasites. Med/Sci 16,122-127). More particularly, PP1/PP2A would play a crucial role in theregulation of anti-apoptotic Bcl-2 proteins and cell survival (Garcia Aet al., PP1 et PP2A des ser/thr phosphatases au coeurde l'apoptose(2001). Med/Sci 17, 1214-1416; Ayllón, V. et al. (2000). Proteinphosphatase 1—is a Ras-activated Bad phosphatase that regulates IL-2deprivation-induced apoptosis. EMBO J. 19, 2237-2246, Ayllón, V. et al.(2000) Bcl-2 targets protein phosphatase 1 alpha to Bad. J. Immunol. 15;166:7345-7352). Identification of peptides that interact with PP2A couldhelp to produce new drugs susceptible to block, by competitiveinhibition, cellular mechanisms induced by viral or parasitic proteins,by their interaction with PP2A and, in particular, the mechanisms ofinfection, pathogen proliferation and neoplastic cell transformation.

In particular, PP2A activation after interaction with the E4orf4adenoviral protein induces apoptosis in transformed cells (Shtrichman R,et al. (2000) Oncogene. 19, 3757-3765). This specific effect requires aninteraction with the B alpha (Bα) subunit of PP2A (Marcellus et al. JVirol. (2000) 74:7869-7877)/Goedert et al. J. Neurochem (2000)75,2155-2162)). All of the above-mentioned observations suggest thehypothesis that the interaction of peptides mimicking the ABC1 and/or A3site with PP2A could lead to apoptosis of transformed cells.

WO9801563 and WO0104329 A1 describe the human E4orf4 protein and itsrole in induction of apoptosis in tumour cells, particularly when thisprotein is expressed via an adenoviral vector. WO0104629 A1 relates tothe modulating and mimetic polypeptides E4orf4 and PP2A, capable toinduce a selective cell death. This document discloses an inventionwhich relates to the ability of the E4orf4 adenoviral protein to inducethe death of neoplastic cells but not of non neoplastic cells. Moreover,WO9801563 A2 relates to the E4orf4 and E4orf6 adenoviral proteins,destined to induce cell death. Finally, the patent application No. FR0110139 describes peptidic compounds that can bind to PP2A.

Also, the Bcl-2 protein family which, in mammals, comprises about twentymembers, can be divided into three sub-families including:

-   -   the anti-apoptotic members (of the Bcl-2 type itself) which all        present at least four conserved motifs, called “BH1 to BH4” for        “Bcl-2 Homology domain”, are necessary to the function of cell        survival. The BH4 motif comprises the interaction domain with        the Raf and Apaf-1 proteins, and with calcineurine;    -   the pro-apoptotic members of the Bax type do not present a BH4        domain; and    -   the pro-apoptotic members of the Bad type only present one BH3        domain.

Mutagenesis experiments show that the anti-apoptotic activity of Bcl-2requires its phosphorylation at the specific residue serine 70 that,when replaced by alanine, inhibits survival. Moreover, the work from Dr.May's team (USA) (2001, Vol. 15, No. 4, pp 515-522) suggests that, inthe presence of IL-3, PP2A can transitorily associate itself with Bcl-2.The use of a point mutant (wherein one alanine residue replaces theserine 70) indicates that the binding of PP2A to Bcl-2 requires thepresence of serine 70 which, consequently, could belong to the bindingsite. The authors thus suggest a dynamic regulatory mechanism wherebyPP2A would be a Bcl-2 phosphatase, antagonistic to Bcl-2 kinases, forexample the PKCs (for discussion, see Garcia A. et al., PP1 et PP2A desser/thr phosphatases au Coerde I'apoptaose (2001). Med/Sci 17,1214-1216).

The presence of these Bcl-2 peptides inside the cell could thus regulatethe phosphorylation, consequently the activity, of Bcl-2 which in turncould block the development of Bcl-2-dependent tumours.

There is thus a need, at the level of antitumour, antiviral andantiparasitic treatments, for peptides derived from E4orf4 and Bcl-2sequences which bind to PP2A or one of its subunits.

SUMMARY OF THE INVENTION

The present invention takes an interest in E4orf4 and Bcl-2 peptides ofsmall dimension that bind a PP2A holoenzyme or one of its sub-units.Contrary to E4orf4 and Bcl-2 native proteins or polypeptidic domains ofgreater dimensions, peptides of small dimensions have the advantage ofbeing easily synthesized, by chemical way or in cellular systems, with ahigh yield and a reduced cost. Peptides of the invention present,moreover, the advantage of being more accessible and of being moreeasily transferred in the cytoplasm or in the nucleus of the cells usingappropriate vectors, for a therapeutic use.

The invention follows from the identification of E4orf4 and Bcl-2peptides, which have a dimension less than 30 amino acids, andespecially peptides having a dimension less than 20 amino acids,interacting in vitro with a purified phosphatase 2A protein holoenzymeor one of its subunits.

In particular, the inventors have identified by the “SPOT synthesis”technique described by Frank and Overwing (Methods in Molecular Biology,1996, vol. 66: 149-169, Epitope Mapping Protocols, G.E. Morris HumanaPress Inc., Totowa, N.J.) the binding sites of the E4orf4 (canineadenovirus type 2) and Bcl-2 proteins interacting with a PP2A holoenzymeor one of its subunits.

On the one hand, the peptides of the invention are peptides having adimension less than 30 amino acids, interacting in vitro with a purifiedPP2A holoenzyme or one of its subunits, said peptides being derived fromthe E4orf4 protein (canine adenovirus type 2) and Bcl-2 protein.Antagonists derived from these peptides and selected because theyinhibit the interaction of viral or parasitic proteins with a specificPP2A holoenzyme could thus constitute new antitumor, antiviral orantiparasitic agents

On the other hand, the invention also concerns a peptide comprising a 12amino acid sequence of the Theileria parva Ckα 2 protein (FD6) capableof interacting with the PP2A sub-unit A and to penetrate in a Hela cell(Garcia, A. et al. (2000). The invention is further concerned with thesequence corresponding to the interaction site of the PP2A with the Basub-unit of the PP2A 1-B site (cf. table 1). This peptide couldpenetrate in cells and, like the E4orf4 protein of the human adenovirus,could interact with PP2A and provoke apoptosis in cancer cells withoutaffecting the normal cells.

The identification method described in application FR no. 0110139 filedon Jul. 27, 2001 in the name of the Applicant, comprises the followingsteps which consist of:

-   -   a) spotting, on a support, E4orf4 or Bcl-2 peptides which there        sequence derived respectively from the viral E4orf4 protein or        the cellular Bcl-2 protein, each spot corresponding to a deposit        of a peptide with a defined sequence,    -   b) contacting the solid support with a solution containing a        phosphatase 2A holoenzyme or one of its subunits under        conditions allowing said peptides present onto the support, to        bind the holoenzyme or one of its subunits, and,    -   c) identifying on the solid support the E4orf4 peptide or the        Bcl-2 peptide onto which said phosphatase 2A holoenzyme or one        of its subunits is bound.

According to step a), different peptides are spotted on a support atdefined positions (spot), each position corresponding to a specificpeptide sequence and the whole then forming a two dimension peptidearray.

Different preparation methods of such arrays have been recentlydescribed (for review, see Figeys et Pinto, 2001 Electrophoresis 22:208-216; and Walter et al., 2000 Curr Opin Microbiol 3: 298-302). Thesemethods comprise in general the covalent binding of peptides on asupport, in particular with the aid of chemical linkers. For instance,one skilled in the art could refer to the “SPOT synthesis” techniquewhich consists of directly synthesize on a cellulosic membrane, peptidescomprising up to 20 residues (Frank and Overwing, Methods in MolecularBiology, 1996, vol. 66: 149-169, Epitope Mapping Protocols, G.E. MorrisHumana Press Inc., Totowa, N.J.).

Generally any method could be used from the moment that such a methodallows the production of such a peptide array, E4orf4 or Bcl-2, spottedon a solid support, useful for detecting specific interaction betweenspotted peptides and the PP2A holoenzyme or one of its subunits.

The totality of the spotted E4orf4 and Bcl-2 peptide sequences cover thecomplete sequence of the corresponding viral or cellular protein.Therefore, the process allows to test in a single step the completesequence, the latter being <<split>> into a number of defined peptides,generally of overlapping sequences.

The spotted peptides have a dimension less than 20 amino acids, andpreferably, less than 15 amino acids.

The peptides may also be spotted on a cellulosic membrane.

The array thus obtained is contacted at step b), with a phosphatase 2Aprotein holoenzyme or one of its subunits.

By “phosphatase 2A protein holoenzyme”, it is meant any dimeric (AC) orheterotrimeric (ABC) complex, purified from a cellular extract orreconstituted following purification of the two subunits (A) and (C) ofa phosphatase type (2A) protein or, if necessary, from the subunit (B),the phosphatases type (2A) proteins are preferably derived from mammals.

The supports are incubated, for instance, in a buffered solutioncomprising the purified phosphatase proteins or one of their purifiedsubunits. A usable buffered solution is TBS (TRIS BUFFER SALINE)containing 5% of skim milk powder and 3% of BSA.

The peptide onto which the phosphatase 2A protein holoenzyme is bound,is identified generally by direct or indirect labelling of thephosphatase protein and identification of spots where the labelledprotein is bound. The binding of the PP2A or one of its subunits on oneof the peptidic spots may be shown, in particular, with antisera,according to known techniques with regards to Western Blot or ELISA,following the incubation of the support containing the peptide arraywith an antibody raised against the subunits (A) or (B) or (C) or amixture of antibodies raised against the PP2A subunits (A), (B) or (C).

The use of the <<SPOT synthesis>> method described in FR no. 0110139 haslead to the identification E4orf4 and Bcl-2 peptides, useful especiallyin anti-tumor, antiviral and antiparasitic treatments, having adimension less than 30 amino acids, even less than 20 amino acids, thesepeptides being able to bind in vitro a phosphatase 2A protein holoenzymeor one of its subunits.

The invention is thus concerned with a E4orf4 or Bcl-2 peptide, naturalor synthetic, with a dimension less than 30 amino acids, preferably ofdimension less than 20 amino acids, characterized in that said peptidespecifically binds in vitro a phosphatase 2A protein holoenzyme or oneof its subunits. By specifically bind, it is understood that the peptideis able to inhibit in a competitive way the binding of a viral orparasitic protein with PP2A peptides.

The invention is also concerned with a peptide comprising a 12 aminoacid sequence of the Theileria parva Ckα 2 protein (FD6) capable ofinteracting with the PP2A sub-unit A and to penetrate in a Hela cell(Garcia, A. et al. (2000) Inhibitions de processus tumoraux ouinfectieux par transfert intracellulaire de peptides mimant des sitesd'interaction avec la serine/threonine phosphatase PP2A). The inventionis further concerned with the sequence corresponding to the interactionsite of the PP2A with the Bα sub-unit of the PP2A 1-B site (cf. table1). This peptide could penetrate in cells and, like the E4orf4 proteinof the human adenovirus, could interact with PP2A and provoke apoptosisin cancer cells without affecting the normal cells.

The identified E4orf4 and Bcl-2 peptides, and the proposed FD6-E4orf4peptides are particularly useful in the treatment of certain tumors,viral or parasitic infections. A person of the art may select, with theaid of competition binding assays, novel peptides, derived fromidentified sequences of the invention, such peptides inhibiting in acompetitive way the binding of the native protein to which they derivewith a PP2A holoenzyme or one of its subunits.

Thus, the invention also concerns a natural or synthetic peptide, asdefined above, characterized in that it inhibit in a competitive way,the interaction of the native protein to which it derives with a PP2Aholoenzyme or one of its subunits.

The peptides of the invention, on order to be efficient in vivo in thetreatment of certain tumors or certain viral or parasitic infections,may be coupled to a vector capable of transferring said peptide in aeukaryotic cell. The small-dimensioned peptides of the invention allowthem to pass through the cell membrane. The use of appropriate vectorsfurther allow to target certain tissue-related peptide, certain cells,even certain specific cellular compartments, and particularly, thecytoplasm or the cell nucleus, in accordance with the desiredtherapeutic effect.

The invention is naturally concerned with the means allowing synthesisof the peptides of the invention. Particularly, the invention isconcerned with a polynucleotide characterized in that its sequence codesfor a peptide of the invention. Preferred polynucleotides arepolynucleotides which their sequence is chosen from one of the followingsequences:

E4 orf4 (375 bp) atggcccacc atcgtctgcc ccgcgtttgt gtaaagggca ttattcattttgaggaagat  60 tttgttagag agcttggatc aatgctggag tctcctatgg agtttctcttcgacaccatt 120 gatgatgtta ctgcttctat attttgtgaa agcatgttta aggctgttgataaaaacaag 180 cctgggatta cttttaaggt ggtcttttac tctcagcttg gttttgagtatgatgatgct 240 cttgcacatt ttaaaggcac tttaattaaa gaaatttctg acgttgttaataatcaccct 300 aatgtaaaca atgcttttag aggaagggag attgtcactg tatctttgttagaagtgttt 360 agtttttgtt cataa BcI-2 (618 bp) atggcgcacg ctgggagaacggggtacgac aaccgggaga tagtgatgaa gtacatccat  60 tataagctgt cgcagaggggctacgagtgg gatgcgggag atgtgggcgc cgcgcccccg 120 ggggccgccc ccgcaccgggcatcttctcc tcccagcccg ggcacacgcc ccatccagcc 180 gcatcccgcg acccggtcgccaggacctcg ccgctgcaga ccccggctgc ccccggcgcc 240 gccgcggggc ctgcgctcagcccggtgcca cctgtggtcc acctggccct ccgccaagcc 300 ggcgacgact tctcccgccgctaccgcggc gacttcgccg agatgtccag ccagctgcac 360 ctgacgccct tcaccgcgcggggacgcttt gccacggtgg tggaggagct cttcagggac 420 ggggtgaact gggggaggattgtggccttc tttgagttcg gtggggtcat gtgtgtggag 480 agcgtcaacc gggagatgtcgcccctggtg gacaacatcg ccctgtggat gactgagtac 540 ctgaaccggc acctgcacacctggatccag gataacggag gctgggtagg tgcatctggt 600 gatgtgagtc tgggctga

It may be advantageous to synthesize a polypeptide comprising therepetition of the identified peptide motifs of the invention.Consequently, the invention concerns a polynucleotide characterized inthat it consists of a multimer of said polynucleotide coding for apeptide of the invention. The invention also concerns a polypeptidecharacterized in that it is obtained from the repetition of a peptide ofthe invention.

The invention is further concerned with a cellular expression vector,characterized in that it comprises a polynucleotide as defined above andregulatory sequences allowing expression of a peptide of the inventionin a host cell.

The invention is also concerned with a preparation method of a peptideaccording to the invention, comprising the transformation of a cell withthe aid of a cellular expression vector as defined above, followed bythe culture of the transformed cellular host, and the recovery of thepeptide in the culture media.

The invention is further concerned with a purified monoclonal orpolyclonal antibody characterized in that it is able to specificallybind to a peptide of the invention.

Antibodies specifically raised against identified peptides of theinvention are obtained, for instance, by the immunization of an animalby injecting a peptide of the invention, and the recovery of theproduced antibodies. A monoclonal antibody may be obtained according tomethods known by one skilled in the art, such as the method described byKohler and Milstein (1975).

The obtained antibodies, specifically raised against phosphatase 2Aprotein targets find their use in particular in immunotherapy. They can,for instance, serve as antagonists of viral or parasitic proteins raisedagainst the phosphatase 2A protein so as to block the viral or parasiticdevelopment.

Furthermore, the polynucleotides coding the peptides of the inventionmay be directly transferred in the nucleus of the target cells, ifnecessary with the aid of appropriate vectors, so as to allow the invivo expression of corresponding peptides, said peptides being likelyable to block by competitive inhibition a specific interaction betweenthe phosphatase 2A protein and the viral or cellular protein which theyderive from.

Thus, the invention is concerned with a pharmaceutical compositioncomprising one of the elements chosen from a polynucleotide of theinvention or an antibody of the invention.

The invention also concerns a pharmaceutical composition comprising oneof the peptides of the invention in combination with a pharmaceuticallyacceptable vehicle.

Moreover, the invention is concerned with the use of a peptide of theinvention, in the preparation of a medicament in the treatment of aviral or parasitic infection.

The peptides of the invention may be advantageously chosen so as tostimulate the induction of apoptosis linked to the activation of thecellular phosphatase 2A protein. Thus, the invention also concerns theuse of a peptide of the invention in the preparation of a medicamentsuited to induce apte apoptosis in target cells, such as tumoral cells.

Cancer results in the specific expression of proteins which theiractivity is regulated by the sequences of the peptides of the invention.The sequences coding the peptides of the invention may be used as probefor detecting, in a specific manner, from RNA extracts of a patient'sbiological sample, the tumoral development.

Furthermore, an antibody of the invention may be used for specificallyrecognizing peptidic sequences contained in viral or cellular proteinsexpressed during the tumoral development.

Thus, the invention is concerned with the use of a polynucleotide of theinvention or an antibody of the invention in the in vitro diagnosis ofcancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Identification of binding sites of E4orf4 protein with PP2A.

FIG. 2: Identification of binding sites of Bcl-2 protein with PP2A.

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the invention, the peptide of theinvention is characterized in that it consists of a fragment of a E4orf4viral protein, a fragment of a Bcl-2 cellular protein or a fragment ofthe proposed Ckα2-E4orf4 peptide, said protein binding in vitro thephosphatase 2A protein, or a sequence distinguishable from said proteinfragment by amino acid substitution or deletion, said sequence retainingthe binding properties to the phosphatase 2A protein holoenzyme or oneof its subunits.

In particular, a distinct sequence is a peptide sequence increasing thebinding affinity to the phosphatase 2A protein holoenzyme or one of itssubunits compared with the native sequence to which said distinctsequence derives. Another distinct sequence as defined above is apeptide sequence homologous to a peptide sequence originally identified,i.e. a sequence derived from a protein of another species than the oneof the peptide sequence originally identified, and of which the primarysequence can be aligned with the peptide sequence originally identifiedwith the aid of an optimal alignment program commonly used, such asBESTFIT (Wisconsin Genetics Software Package, Genetics Computer Group,GCG). In particular, a sequence A will be considered as being homologousto a sequence B if the sequences A and B present a homology of at least50%, preferably 75%, after alignment of the sequences with the aid of aprogram such as BESTFIT. More preferably, two sequences are alsoconsidered homologous if the sequence are almost identical with theexception of some residues which may represent 10 to 20% of variabilityover the total sequence. Moreover, similar amino acids having similar bytheir chemical function (such as Arg and Lys) are considered asequivalents.

A particular preferred peptide of the invention is a fragment of theadenovirus type 2 E4orf4 protein, and more specifically a fragment ofthe canine adenovirus type 2 E4orf4 protein, or a sequencedistinguishable from said protein fragment by amino acid substitution ordeletion, said sequence retaining the binding properties to thephosphatase 2A protein holoenzyme or one of its subunits.

In a more preferred embodiment, a peptide of the invention ischaracterized in that it includes one of the following sequences:

A) RELGSMLE SPMEFLFDTI DDVTAS (SEQ. ID NO: 1) b) LGSMLE SPMEFLFDTIDDVTAS (SEQ. ID NO: 2) c) SMLE SPMEFLFDTI DDVTAS (SEQ. ID NO: 3) d)HFKGTLIKEISDVVNN (SEQ. ID NO: 4) E) AFRGRE VTVSLLEVFSFC (SEQ. ID NO: 5)f) VKKKKIKREIKI SMLE SPMEFLFDTI D (SEQ. ID NO: 6) DVTAS g)ARTSPLQTPAAPGAAAGPAL (SEQ. ID NO: 7) h) RFATVVEELFRDGVNW (SEQ. ID NO: 8)i) RPLFDFSWLSLKTLLSLALVGA (SEQ. ID NO: 9) j) PLQTPAAPGAAAGPAL (SEQ. IDNO: 10) k) LFDFSWLSLKTLLSLALVGA (SEQ. ID NO: 11) l) a sequencedistinguishable from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 byamino acid subtitution or deletion, said sequence retaining the bindingproperties to the phosphatase 2A pro- tein holoenzyme or one of itssubunits.

Among the peptide sequences distinguishable from SEQ ID NO: 1, 2, 3, 4,5 or 6 by amino acid substitution or deletion, and being contemplated bythe present invention, are more particularly the peptides which theirsequence is enclosed in one of the sequence of the E4orf4 protein,different variants of canine and human adenovirus type 2, andcorresponding to the homologous sequences of these variants of SEQ IDNO: 1, 2, 3, 4, 5 or 6.

Among the peptide sequences distinguishable from SEQ ID NO: 7, 8, 9, 10or 11 by amino acid substitution or deletion, and being contemplated bythe present invention, are more particularly the peptides which theirsequence is enclosed in one of the sequence of the Bcl-2 protein, andcorresponding to the homologous sequences of these variants of SEQ IDNO: 7, 8, 9, 10 or 11.

A preferred peptide according to the invention is a peptide chosen fromone of the sequences SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 andcharacterized in that its administration induces apoptosis of cells, inparticular apoptosis of tumoral cells.

The invention preferably aims at the use of a peptide which its sequencederives from a fragment of the E4orf4 protein, as defined above, in thepreparation of a medicament suited to inhibit a viral infection.

Examples

PP2A Purified Proteins

The trimeric PP2A1 protein has been purified to homogeneity from porcinebrain.

Recombinant protein A (structural sub-unit of PP2A) expressed inbacteria.

Recombinant protein B55 (regulatory sub-unit of PP2A) expressed inbacteria.

Identification of E4orf4 and Bcl-2 Binding Sites with PP2A

We have mapped the binding sites between the E4orf4 proteins (coded bythe canine adenovirus type 2) and the anti-apoptotic Bcl-2 protein withPP2A by using the <<peptides spot>> technique previously described(Frank et Overwing. (1996). Meth. Mol. Biol. 66, 149-169).

This approach is based on the use of membranes containing thereondodecapeptides representing the whole protein of interest (E4orf4 andBcl-2) with a two amino acid gap per peptide.

Each membrane is first saturated one hour at room temperature with TBScontaining 5% skim milk powder and 3% BSA, then incubated overnight inthe same buffer in the of 4 μg/ml of purified protein (sub-unit A ofPP2A and PP2A1 holoenzyme): Fifty six dodecapeptides covering the wholesequence of the E4orf4 protein and hundred-fifteen dodecapeptidescovering the whole sequence of the Bcl-2 protein were synthesized andcovalently bound to cellulosic membranes.

E4orf4: MAHHRLPRVCVKGIIHFEEDFVRELGSMLESPMEFLFDTIDDVTAFTSIFCESMFKAVDKNKPGITFKVVFYSQLGFEYDDALAHFKGTLIKEISDVVNNHPNVNNFTAFRGREIVTVSLLEVFSFCS. Bcl-2:MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAFTPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLALRQAGDDFFTSRRYRGDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNFTREMSPLVDNIALWMTEYLNRHLHTWIQDNGG WVGASGDVSLG.

The specific interaction of each purified protein (respectively thestructural sub-unit A, the regulatory sub-unit of B55(B) or the trimericABC holoenzyme (named PP2A1) with a peptide sequence is shown by westernblot, following incubation of the membrane with an antibody raisedagainst the structural protein A (A), the regulatory sub-unit B (B) andwith a pool of antibodies recognising the A, B, and C proteins of PP2A(ABC) (FIGS. 1 and 2).

Example 1

Identification of Peptidic Sequences Containing Binding Sites of theE4orf4 Protein Coded by the Canine Adenovirus Type 2 with Proteins ofthe PP2A Family.

Screening of a membrane containing peptides covering the E4orf4sequences with different forms of purified PP2A (FIG. 1) allowed us toidentify five amino acid sequences containing interaction sites ofE4orf4 with PP2A (cf. table 1).

We were able to respectively determine:

a peptidic sequence containing a binding site of E4orf4 with thestructural sub-unit B (“site B1” corresponding to peptides 12 to 18).

Three peptidic sequences corresponding to three binding sites of E4orf4with the sub-unit A (“site A 1” corresponding to peptides 13 to 18 and“site A2” corresponding to peptides 41 to 43 and “site A3” correspondingto peptides 52 to 55).

Two peptidic sequences corresponding to two binding sites of E4orf4 withthe PP2A1 protein (“site B1” corresponding to peptides 14 to 18 and“site A3” corresponding to peptides 52 to 55).

It is interesting to note that (table 1) the B1, A1 and ABC1 sitespartially overlap one another thus suggesting:

that the sub-units A and B can interact on the same site which has neverbeen established in the PP2A system. Moreover, the interaction of thetrimeric PP2A with this site requires a shorter sequence thus suggestinga conformational regulation.

TABLE 1 Binding sites of E4orf4 with different PP2A proteins Site B123-RELGSMLE SPMEFLFDTI DDVTAS-46 Site A1 25-LGSMLE SPMEFLFDTI DDVTAS-46Site ABC1 28-SMLE SPMEFLFDTI DDVTAS-4 Site A2 83-HFKGTLIKEISDVVNN-98Site A3 106-AFRGRE VTVSLLEVFSFC-124

Example 2

Identification of Peptidic Sequences Containing Binding Sites of Bcl-2Protein with Proteins of PP2A Family.

Screening of a membrane containing peptides covering the Bcl-2 sequenceswith different forms of purified PP2A (FIG. 2) allowed us to identifyfive amino acid sequences containing interaction sites of Bcl-2 withPP2A (cf. table 2).

We were able to respectively determine:

a peptidic sequence containing a binding site of Bcl-2 with theregulatory sub-unit B (“site 1-B” corresponding to peptides 33 to 37).

two peptidic sequences containing two binding sites of Bcl-2 with thestructural sub-unit A (site A1 corresponding to peptides 64-67 and siteA2 corresponding to peptides 104-109).

two peptidic sequences containing a binding site of Bcl-2 with the PP2A1holoenzyme (site 1-ABC corresponding to peptides 35 to 37 and site 2-ABCcorresponding to peptides 105-109).

It is interesting to note that the 1-ABC and 2-ABC sites correspondrespectively to the 1-B and A2 sites with respectively two and fouramino acid gaps probably linked to a different conformation thusresulting in the interaction of proteins A and B within the holoenzyme.

Moreover it is noteworthy that the 1-B/1-ABC site is located at theser-70 level where the phosphorylation regulated the PP2A activity whilethe 2-ABC/A2 site is located at the C-terminus end of the protein.Contrary to the interaction sites with PP1, these two sites do notinterfere with the BH domains of PP2A (see Garcia A. et al. PP1 et PP2Ades ser/thr phosphatases au coeur de I'apoptose (2001). Med/Sci 17,1214-1216 for general discussion).

TABLE 2 Binding sites of Bcl-2 with different PP2A proteins Site 1-B67-ARTSPLQTPAAPGAAAGPAL-86 Site A1 129-RFATVVEELFRDGVNW-144 Site A2207-RPLFDFSWLSLKTLLSLALVGA- 228 Site 1-ABC (site 1B)71-PLQTPAAPGAAAGPAL-86 Site 2-ABC (site A2) 209-LFDFSWLSLKTLLSLALVGA-228

1-23. (canceled)
 24. An isolated peptide that is at least 90% identicalwith a complete amino acid sequence of VKKKKIKREIKI SMLE SPMEFLFDTIDDVT. (SEQ. ID NO: 6) AS.


25. A composition comprising the isolated peptide of claim 24 and apharmaceutically acceptable carrier.
 26. A composition comprising theisolated peptide of claim 24 coupled to a vector which can transfer saidpeptide into a eukaryotic cell.
 27. The isolated peptide of claim 24,wherein the peptide specifically binds a phosphatase 2A proteinholoenzyme or one of its subunits in vitro.